Extended Data Fig. 3: Functional Flag-tagged RcaT-RT co-immunoprecipitate independently of temperature.
From: Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems
a–b, Volcano plots of affinity purifications of 3xFlag-tagged RrtT (panel a) and RcaT (panel b) at 37 °C and 20 °C in wild-type and different mutant backgrounds. Same experiment as in Fig. 2a, b, presenting additionally the AP data from 37 °C. The x-axis represents the average log2 ratio of identified proteins (by more than 2 independent peptides) in rrtT-3xFlag and rcaT-3xFlag AP samples compared to an untagged STm WT control strain (n = 2 biological), and the y-axis represents p values of these log2 ratios (two-tailed limma). c, Flag-tagged RcaT retains its function. Untagged and rcaT-3xFlag tagged STm strains (WT and retron-deletions) were grown for 5-6 h at 37 °C in LB, serially diluted, spotted on LB plates, and incubated at indicated temperatures. * denotes ΔSTM14_4645::cat, used to co-transduce the scarless Flag-tagged rcaT (co-transduction verified by PCR) (n = 2 biological). d, Flag-tagged RrtT retains its function. Untagged and rrtT-3xFlag tagged STm strains (WT and retron-deletions) were grown for 5-6 h at 37 °C in LB, serially diluted, spotted on LB plates, and plates were incubated either at indicated temperatures (n = 2 biological). e, Flag-tagging rrtT does not alter retron protein expression, whereas flag-tagging rcaT decreases levels of both retron proteins. Proteins in input (whole proteome) samples used for samples shown in Fig. 2a, b were quantified by MS. Protein levels (log2-fold change) of RcaT and RT were compared between Flag-tagged and untagged STm WT strains (x-axis). Lines denote the mean and come from two biological replicates. RT was downregulated in rcaT-3xFlag tagged strains, whereas RcaT was not even detected (n.d.), presumably due to lower levels (chromosomal RcaT is at the level of detection by MS, so small changes can bring it below detection). Note that despite lower levels of both retron components, rcaT-3xFlag retains its function (panel c) and is expressed (Extended Data Fig. 2d).
